ABSTRACT: In cancer, aldehyde dehydrogenase (ALDH) enzyme activity is high in dendritic cells and regulatory T-cells, hypothesized to modulate the immune response and also in cancer stem cells (CSCs) to aid the development of recurrent resistant disease. It is therefore surprising that drugs targeting ALDH have not been developed to modulate these processes in cancer. To investigate this issue, we tested the efficacy of existing isoform-specific and broad-spectrum ALDH inhibitors on cultured cells and found that none were very effective at modulating cell survival. Furthermore, we predicted that a broad-spectrum inhibitor would be needed due to the overlapping functions of the ALDH family members. These observations provided the rationale to develop a non-toxic broad- spectrum ALDH inhibitor, which would kill ALDH+ CSCs to prevent recurrent resistant disease, and improve immunotherapy of melanoma. Thus, the central hypothesis is that a non-toxic ALDH inhibitor can be developed to reduce recurrent resistant disease development mediated by ALDH+ CSCs and improve the efficacy of existing immunotherapies by reducing immune suppression. Preliminary data show development of a potent non-toxic ALDH inhibitor built on the backbone of the Isatin natural product. This agent eliminates CSCs with high ALDH activity, preventing increases in ALDH+ cells typically seen with traditional drug treatments. Finally, the inhibitor improves the preclinical efficacy of anti-PD1 immunotherapy in a syngeneic mouse melanoma model where this approach alone is not effective. Based on these hypothesis-supporting preliminary data, the following Specific Aims are proposed. First, determine how effectively a non-toxic broad-spectrum ALDH inhibitor eliminates the melanoma ALDH+ CSC population that expands when treating with traditional chemotherapy. Furthermore, determine its potency when combined with traditional chemotherapy to prevent recurrent resistant disease development. This will be achieved by isolating ALDH+ and ALDH- cell populations from cell lines and patient derived (PDX) tumors and determining inhibitor efficacy for preventing recurrent resistant disease development when provided in combination with current clinically relevant chemotherapy. Finally, the mechanistic basis for inhibition of resistance by the agent combination will be identified. Second, determine the efficacy of a non-toxic broad-spectrum ALDH inhibitor on immune regulatory and effector cells in the tumor microenvironment, alone and when combined with anti-PD1 antibody immunotherapy. Specific targeting of ALDH in melanoma tumor cells and in host cells on therapeutic efficacy will be tested. Mechanisms leading to enhanced immune response will be identified by defining the impact of the inhibitor and antibody combinations on immune cell composition and function within the tumors, assessing the relative ALDH levels in the immune cell populations, and using transient and genetic approaches to target the modulating immune cells. These significant discoveries would demonstrate the efficacy of targeting ALDH enzymes in cancer for modulating resistance and immune cell function, providing needed preclinical validation necessary for clinical translation.